N-Acylhydrazones as inhibitors of PDE10A

Article abstract of DOI:10.1016/j.bmcl.2011.05.100

Cyclic nucleotide phosphodiesterases (PDEs) are represented by a large superfamily of enzymes. A series of hydrazone-based inhibitors was synthesized and shown to be novel, potent, and selective against PDE10A. Optimized compounds of this class were efficacious in animal models of schizophrenia and may be useful for the treatment of this disease.

Full text of DOI:10.1016/j.bmcl.2011.05.100

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4155–4159
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Acylhydrazones as inhibitors of PDE10A
Jennifer L. Gage a, Rene Onrust a, Derek Johnston b, Andrew Osnowski b, Wendy MacDonald b,
Lee Mitchell b, László Ürögdi c, Alex Rohde a, Kevin Harbol a, Sasha Gragerov a,
György Dormán c, Tom Wheeler a, Vince Florio a, Neil S. Cutshall a,
⇑
a Omeros Corp., 1420 Fifth Ave., Suite 2600, Seattle, WA 98101, USA
b Scottish Biomedical, Telford Pavilion, Todd Campus, West of Scotland Science Park, Glasgow, Scotland, UK
c AMRI Hungary Zrt., Záhony. u. 7, 1031 Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic nucleotide phosphodiesterases (PDEs) are represented by a large superfamily of enzymes. A series
of hydrazone-based inhibitors was synthesized and shown to be novel, potent, and selective against
PDE10A. Optimized compounds of this class were efficacious in animal models of schizophrenia and
may be useful for the treatment of this disease.
Received 7 April 2011
Revised 24 May 2011
Accepted 25 May 2011
Available online 2 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
PDE10
PDE10A
Phosphodiesterase
Schizophrenia
Hydrazide
PDE
Enzyme inhibitor
Hydrazone
Acylhydrazone
Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of
enzymes that hydrolyze the second messengers cyclic adenosine
and cyclic guanosine 3’,5’-monophosphate (cAMP and cGMP). Inhi-
bition of PDEs has been an active area of drug discovery.1 Selective
inhibitors of the individual PDE families have widely been sought
for a broad indication of therapeutic uses. Potential therapeutic uses
include the treatment of allergies,2a COPD,2b persistent pulmonary
hypertension of the newborn,2c carcinoma,2d angina,2e cardiovascu-
lar regulation,2f depression,2g and erectile dysfunction.2h PDE10A
was identified independently by three groups in 1999 and shows a
dual activity of hydrolysis of both cAMP and cGMP.3–5 PDE10A is
an attractive target because it may offer a novel therapy for the treat-
mentof neurologicaland psychiatricdisordersincludingParkinson’s
disease, Huntington’s disease, schizophrenia, and disorders that
would benefit from increasing levels of cAMP and cGMP within neu-
rons.6 Increasing neuronal cAMP and cGMP may benefit patients
with neurological and psychiatric disorders through striatal activa-
tion and behavioral suppression.
There have also been published reports of X-ray and computational
data obtained for PDE10 complexed with ligands.7–9 In an effort to
identify inhibitors of PDE10A, a high-throughput screening cam-
paign was conducted and provided screening hits which contained
an N-acylhydrazone linker typified by potent screening hit 1
(Fig. 1). Hydrazone-based molecules have been popular as poten-
tial therapeutics for tuberculosis,10a,10b and as small molecule
inhibitors of numerous targets such as human glucagon recep-
tor,10c and pituitary adenylate cyclase-activating polypeptide
receptor.10d Here we report the structure–activity relationship
(SAR) study of a novel class of highly selective hydrazone-based
small molecules that inhibit PDE10A in the low nanomolar range
N
O
O
S
N
N
O
To date, a number of drug-like small molecules have been re-
ported to inhibit PDE10A, although few have entered clinical trials.
H
1
IC50 0.049 µM
⇑
Corresponding author. Tel.: +1 206 676 0804; fax: +1 206 676 0808.
E-mail address: ncutshall@omeros.com (N.S. Cutshall).
Figure 1. Schematic representation of 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.100

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J. L. Gage et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4155–4159
Table 1
O
O
Dimethoxyphenyl region SAR of substituted N-acylhydrazones
Ar1
Ar2
=
=
N
Ar1
Ar2
S
N
a
b
N
N
R3
5
Ar Cl
R2
R4
R5
O
O
4
O
S
N
N
N
H
R1
R6
6; R1 =H
7; R1 = Me
R2
R3
R4
R5
R6
PDE10A IC50 (lM)
a
Compd
Scheme 2. Reagents and conditions: (a) methyl thioglycolate, N,N-diisopropyleth-
ylamine, DMF; (b) glycine methylester HCl or sarcosine methylester HCl, Et3N,
EtOH, reflux.
1
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
H
H
H
H
H
H
H
OMe
OMe
H
OMe
H
OMe
H
Cl
F
Br
Me
CN
CF3
OCHF2
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
CN
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
F
H
H
H
H
H
H
H
H
H
0.049
1.2
1.4
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OCHF2
OEt
0.15
0.087
0.19
0.093
0.10
0.16
0.25
0.31
0.26
0.42
0.62
0.22
1.7
O
O
O
O
a
b
S
Br
SH
S
Br
9
8
10
Scheme 3. Reagents and conditions: (a) methyl bromoacetate, K2CO3, DMF; (b)
PhB(OH)2, Pd(PPh3)4 (5 mol %), Na2CO3, THF/H2O.
CF3
F
Cl
OCF3
O-iPr
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
0.66
0.20
0.077
0.19
4.6
NO2
NO2
NO2
NO2
S-K+
H
H
H
H
a
b
c
O
S
O
NH2
S
14
H
4.8
O
11
12
13
S
OMe
CN
Br
Cl
F
0.010
0.011
0.03
0.045
0.051
0.014
0.0048
Scheme 4. Reagents and conditions: (a) NaNO2, HCl (aq), 5 °C; potassium ethyl
xanthate, 40 °C; (b) KOH, EtOH, reflux; (c) methyl bromoacetate, K2CO3, DMF.
OMe
OMe
Br
Biphenyl analog 49 was synthesized from 4-bromothiophenol 8
by alkylation with methyl bromoacetate to provide 9 followed by
Suzuki coupling to give thioacetate 10 as shown in Scheme 3. Fur-
ther synthetic transformations via the route depicted in Scheme 1
provided compound 49.
Nitronaphthalene analog 52 was synthesized from naphthyl-
amine 11 as shown in Scheme 4. Compound 11 was converted to
xanthate 12 followed by hydrolysis to potassium thiolate 13. Alkyl-
ation with methyl bromoacetate provided precursor 14 which was
used to synthesize analog 52 in two steps.
a
Values were calculated from the average of at least two experiments.
N
N
S
N
S
a
b
O
O
OH
NH2
N
R1
N
N
H
H
R2
2
3
1, 17-44, 46-48,
50-51, 53-56, 59
N-Substituted analog 61 was synthesized as depicted in
Scheme 5. Thus, treatment of aldehyde 15 with methyl hydrazine
provided intermediate 16. Condensation of 16 with the requisite
methyl ester then gave the desired N-methyl analog 61.
Scheme 1. Reagents and conditions: (a) (i) Lawesson’s reagent, THF, 80 °C, 30%; (ii)
methyl bromoacetate, K2CO3, DMF, 88%; (iii) NH2NH2ÁH2O, EtOH, reflux, 80%; (b) R1-
C(O)R2, EtOH, HOAc (cat.), reflux.
and show oral animal efficacy in behavioral models for
schizophrenia.
As part of a lead optimization strategy, acyl hydrazines were
screened in an in vitro enzymatic assay for their ability to inhibit
PDE10A.11 In vitro potency optimization focused on modifying
the three regions of the inhibitor: the heteroaromatic portion
(quinoline), the dimethoxy-phenyl region, and the central linker.
Initial SAR studies focused on modifications to the substituted
phenyl ring while the 3-methyl quinoline and linker regions were
held constant (Table 1). Based on previous reports of PDE10A
inhibitors, it was known that methoxy substituted phenyl groups
could be important recognition elements for enzyme binding.7,9
In order to gain insight into the pharmacophore of screening hit
1, it was modified by removing one of the methoxy groups (17, 18)
and by changing the substitution pattern (19). These modifications
resulted in a 3 to 28-fold loss in potency against PDE10A. Due to
concerns about the metabolic liability of the 4-methoxy group
from in vitro liver microsome studies (data not shown), it was of
interest to search for less labile functionalities. Compounds 20–25
illustrate that replacement of the 4-methoxy group with chloro,
fluoro, bromo, methyl, cyano or trifluoromethyl group led to less
The synthetic route used to synthesize the analogs in Table 1 is
depicted in Scheme 1. Commercially available 2-methylquinolin-
4-ol (2) was treated with Lawesson’s reagent to provide the corre-
sponding thiol which was then converted to intermediate 3 in good
yield. Intermediate 3 was readily condensed with a variety of alde-
hydes or ketones in the presence of catalytic acetic acid to provide
the corresponding acylhydrazones in typically >70% yield. In all
cases only the thermodynamically stable geometrical E-isomer
was detected and isolated. Compounds 17–44, 46–48, 50, 51,
53–56, 59 and the oxygen linked compound, 57, were synthesized
in a similar fashion as outlined above.
In order to access a broader range of synthetic precursors it was
of interest to explore additional chemistry routes. As shown in
Scheme 2, aryl chlorides could readily be reacted with either a
thiolate anion or amine functionality to provide intermediates 5,
6, and 7. Further transformations using previously described condi-
tions provided final compounds 45, 58 and 60, respectively.

J. L. Gage et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4155–4159
4157
N
O
N
S
O
O
O
S
O
O
O
a
O
O
O
O
O
O
N
N
b
N
N
H
15
16
61
Scheme 5. Reagents and conditions: (a) methyl hydrazine, EtOH, reflux; (b) EtOH, HOAc (cat.), reflux.
Table 2
Heteroaromatic region SAR of substituted N-acylhydrazones
O
O
O
R1
O
S
N
N
H
R1
PDE10A IC50
1.07
(
lM)
Compd
R1
PDE10A IC50
0.14
(lM)
a
a
Compd
N
45
51
N
N
CF3
NO2
O
46
0.86
52
0.10
N
N
47
48
49
50
0.66
0.55
0.26
0.19
53
54
55
56
0.024
0.028
0.010
0.008
O
N
Cl
N
N
N
O
a
Values were calculated from the average of at least two experiments.
active analogs. Replacement of the 3-methoxy group also provided
less potent inhibitors as demonstrated by compounds 27–32 com-
pared to parent compound 1. Bis-difluoromethoxy compound 26
PDE10A inhibition was observed with mono-aromatic ring systems
such as 45–48. In contrast, fused bicyclic aromatic ring systems
provided examples with substantially better activity. For instance,
4-substituted quinoline based compounds (53–56) provided the
most potent inhibitors. Examples of 2- and 8-substituted quino-
lines such as compounds 50 and 51 failed to provide significant
improvements in potency and were not pursued. Installation of
other aromatic systems such as biphenyl and naphthyl compounds
49 and 52 also failed to provide necessary improvements in
potency.
The final area of SAR exploration studied modifications to the
hydrazone linker region (Table 3). A comparison of compounds 1
and 59 illustrates that even a relatively small alkyl group substi-
tuted in place of hydrogen at the benzylidene carbon resulted in
approximately a threefold loss of potency. It was also of interest
to determine if the hydrazone nitrogen could tolerate substitu-
tions. A comparison of compounds 55 and 61 illustrates that sub-
stitution of the NH hydrogen with a methyl group also resulted
in almost a threefold loss of potency. Lastly, it was of great interest
to know the effect of replacement of the heteroatom linker. It was
gave an IC50 of only 0.31
the 1- or 6-positions on the phenyl ring (33–37) gave analogs with
IC50 values in the range of 0.077–4.8 M. In contrast, 3,4,5-trisub-
lM. Incorporation of a halogen at either
l
stituted phenyl analogs where R5 was methoxy, cyano, bromo,
chloro or fluoro (compounds 38–42) gave equivalent or better po-
tency against PDE10A as compared to compound 1. Finally,
replacement of the 4-methoxy group of 38 was examined via the
synthesis of 4-cyano derivative 43 and 4-bromo compound 44.
While both gave excellent in vitro activity, compound 44 was both
the most potent example with an IC50 of 4.8 nM and provided im-
proved metabolic stability (data not shown).
A second area of SAR effort focused on modification of the
3-methyl quinoline portion of the molecule. It was determined early
in the course of SAR studies that an aromatic group, and preferen-
tially a functionalized bicyclic ring system, provided potent inhib-
itors against PDE10A. Table 2 shows representative examples of
the substituents explored. In the majority of cases, only modest

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J. L. Gage et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4155–4159
Table 3
Linker modifications
3500
PCP
N
X
R3
PCP + Cpd 56
3000
O
O
N
2500
N
O
R5 R4
2000
R3
R4
R5
PDE10A IC50
(
lM)
a
Compd
X
1
57
58
59
55
60
61
S
O
NH
S
S
H
H
H
H
OMe
OMe
OMe
H
H
H
Me
H
H
H
H
H
H
H
Me
0.049
1.6
8.0
0.18
0.010
0.36
0.026
1500
1000
500
0
NMe
S
H
H
a
Values were calculated from the average of at least two experiments.
INT 1
INT 2
INT 3
INT 4
INT 5
Table 4
Rat pharmacokinetic profile of compound 56
Intervals, 4 min each
PK propertiesa
Value
Figure 3. Compound 56 reduced PCP-induced hyperactivity. Activity was moni-
tored by number of beam breaks in an open field novel environment. Compound
(20 mg/kg) or vehicle (30 mM H3PO4, 0.2% tween 80, 5% DMSO) were administered
IP 20 min before testing, and PCP was injected by the same route 10 min before
testing. Normal activity in this experiment ranges between 500 and 1000. Eight
mice per group were used, t-test for each interval showed p <0.005.
T1/2 (h), iv
11.3
3.3
17.4
0.25
539
21
CL (L/h/kg), iv
Vss (L/kg), iv
Tmax (h), po
Cmax (ng/ml), po
F (%)
a
iv dose: 4 mg/kg, po dose: 9 mg/kg.
only a limited number of non-sulfur based analogs were prepared
and tested.
30
25
20
15
10
5
As part of a lead optimization strategy, acylhydrazones were
screened in in vitro enzymatic assays for their ability to inhibit
PDE10A12 while maintaining selectivity against a broader panel
of PDEs. It was found that compound 56 possessed excellent selec-
tivity for PDE10A against PDE1A-PDE11A (excluding PDE6A which
was not tested). Selectivity ratios varied from 110-fold to >1250-
fold. In no case were any inhibitors found to be more selective
against another PDE as compared to PDE10A.
Selected compounds were also evaluated in pharmacokinetic
(PK) studies in rats (Table 4). In vitro observations indicated that
the compounds had adequate stability in simulated gastic fluid to
be absorbed orally. Compound 56 demonstrated high clearance,
and bioavailability was modest (21%). However, the PK profile
was favorable enough to warrant further evaluation in behavioral
models.
Compound 56 was assessed in two different behavioral models
shown to have predictive validity for schizophrenia. Figure 2
shows that in the conditioned avoidance response (CAR) model13
the compound demonstrated a dose-dependent and statistically
significant reduction upon intraperitoneal (IP) injection at doses
of 10 and 30 mg/kg.
The attenuation of hyperlocomotion induced by phencyclidine
(PCP) has also been shown to have predictive validity for anti-psy-
chotic drugs.14 Figure 3 shows that in this assay, 56 significantly
reduced PCP-induced hyperactivity at a dose of 20 mg/kg (IP).
In conclusion, the first reported synthesis and evaluation of
hydrazone-based inhibitors of PDE10A have been described. SAR
studies led to the discovery of compound 56, an excellent inhibitor
that showed a high degree of enzyme selectivity against other
PDEs. The compound was absorbed orally and demonstrated
in vivo efficacy in two animal behavioral models of schizophrenia.
This compound also showed a positive effect using an in vivo cog-
nition model (data not shown). The findings from this study will be
useful for further optimization and will be the subject of additional
reports in due course.
0
0
10
30
Dose, mg/kg
Figure 2. Attenuation of conditioned avoidance response by 56. Mice were trained
to avoid foot-shock by moving to the other side of a shuttle-box in response to
conditional stimulus (CS). Training proceeded until mice demonstrated at least 25
avoidances out of 30 trials. Compound 56 was administered intraperitoneally in
30 mM H3PO4, 0.2% tween 80, 5% DMSO 20 min before testing. Control group
received vehicle injection. Eight mice per group were used. Paired-sample t-test
was used to compare performance of the same animals on the day of the
experiment and on the previous day when they were injected with the vehicle. p
<0.05 for 10 mg/kg and p <0.001 for 30 mg/kg.
clearly demonstrated by comparison of 1to 57 and 58 that replace-
ment of sulfur with either oxygen or an unsubstituted amino moi-
ety led to a significant loss of inhibition. Attempts to incorporate a
substituted amino moiety into the linker also led to reduced po-
tency as illustrated by compound 60. Based on these observations

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4159
9. Pandit, J. US 2005/0202550 A1.
Acknowledgements
10. (a) Carvalho, S. A.; da Silva, E. F.; de Souza, M. V. N.; Lourenco, M. C. S.; Vicente,
F. R. Bioorg. Med. Chem. Lett. 2008, 18, 538; (b) Mamolo, M. G.; Falagiani, V.;
Zampieri, D.; Vio, L.; Banfi, E. IL Farmaco 2001, 56, 587; (c) Madsen, P.; Ling, A.;
Plew, M.; Sams, C. K.; Knudsen, L. B.; Sidelmann, U. G.; Ynddal, L.; Brand, C. L.;
Andersen, B.; Murphy, D.; Teng, M.; Truesdale, L.; Kiel, D.; May, J.; Kuki, A.; Shi,
S.; Johnson, M. D.; Teston, K. A.; Feng, J.; Lakis, J.; Anderes, K.; Gregor, V.; Lau, J.
J. Med. Chem. 2002, 45, 5755; (d) Beebe, X.; Darczak, D.; Davis-Taber, R. A.;
Uchic, M. E.; Scott, V. E.; Jarvis, M. F.; Stewart, A. O. Bioorg. Med. Chem. Lett.
2008, 18, 2162.
11. PDE10 Assay. Compounds were tested for PDE10 potency by measuring the
inhibition of PDE10 hydrolysis of [3H]cGMP to [3H]GMP. Generally, eight
dilutions of compound were assayed in 50 mM Tris–HCl pH 7.5, 8.3 mM MgCl2,
0.5 mg/mL BSA, 1.7 mM EGTA, 16 nM [3H]cGMP and 1% DMSO. To start the
The authors would like to thank the Stanley Medical Research
Institute for funding.
References and notes
1. Manallack, D. T.; Hughes, R. A.; Thompson, P. E. J. Med. Chem. 2005, 48, 3449.
2. (a) Grootendorst, D. C.; Klaus, R. F. Curr. Opin. Allergy Clin. Immunol. 2002, 2, 61;
(b) Barnes, P. J. Nat. Rev. Drug Disc. 2002, 1, 437; (c) Travadi, J. N.; Patole, S. K.
Pediatr. Pulmonol. 2003, 36, 457; (d) Marko, D.; Pahlke, G.; Merz, K.-H.;
Eisenbrand, G. Chem. Res. Toxicol. 2000, 13, 944; (e) Ghofrani, H. A.; Osterloh, I.
H.; Grimminger, F. Nat. Rev. Drug Disc. 2006, 5, 689; (f) Kass, D. A.; Beavo, J. A.
Circ. Res. 2007, 101, 1084; (g) Menniti, F. S.; Faraci, W. S.; Schmidt, C. J. Nat. Rev.
Drug Disc. 2006, 5, 660; (h) Rotella, D. P. Nat. Rev. Drug Disc. 2002, 1, 674.
3. Soderling, S. H.; Bayuga, S. J.; Beavo, J. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
7071.
reaction, mouse PDE10 (BPS BioSciences, CA) was added to
a final
concentration of 25 ng/ml. The reaction was incubated at 30 °C for 20 min.
PDE10 hydrolysis was terminated by the addition of yttrium silicate beads (GE
Healthcare, RPNQ0150) and counted on
a Wallac Microbeta scintillation
counter 1–2 h following the addition of the beads. Data was analyzed using
XLfit (Microsoft) from which IC50 values were obtained.
4. Fujishige, K.; Kotera, J.; Michibata, H.; Yuasa, K.; Takebayashi, S.; Okumura, K.;
Omori, K. J. Biol. Chem. 1999, 274, 18438.
5. Loughney, K.; Snyder, P. B.; Uher, L.; Rosman, G. J.; Ferguson, K.; Florio, V. A.
Gene 1999, 234, 109.
12. The inhibition of other PDE enzymes by the PDE10A inhibitors was evaluated
under the same conditions described above for PDE10A. Fractional inhibition
was evaluated at four concentrations (0.1, 1, 10, and 100 lM). In cases where
inhibition at the highest concentration was less than 50%, the lower limit value
in the logistic model was fixed to 0% activity.
13. Wadenberg, M. L.; Hicks, P. B. Neurosci. Biobehav. Rev. 1999, 23, 851.
14. Gleason, S. D.; Shannon, H. E. Psychopharmacology (Berl) 1997, 129, 79.
6. Hebb, A. L.; Robertson, H. A. Curr. Opin. Pharm. 2007, 7, 86.
7. Chappie, T. A.; Humphrey, J. M.; Allen, M. P.; Estep, K. G.; Fox, C. B.; Lebel, L. A.;
Liras, S.; Marr, E. S.; Menniti, F. S.; Pandit, J.; Schmidt, C. J.; Tu, M.; Williams, R.
D.; Yang, F. V. J. Med. Chem. 2007, 50, 182.
8. Wang, H.; Liu, Y.; Hou, J.; Zheng, M.; Robinson, H.; Ke, H. PNAS 2007, 104, 5782.